The overall objective of this research program concerns the aspect of a hypothesis that mutations of a DNA replication protein can be a source of genomic instability and an early event in tumorigenesis. Our previous studies have suggested that a specific region of DNA polymerase alpha (Polalpha) plays a significant role in genome stability maintenance. We have also found that absence of flap endonuclease-1 (FEN-1) nuclease activity induces a dominant mutator phenotype characterized by duplication of genomic sequences. In this application, we plan to test the hypothesis that mutations in the specific region of Polalpha and a nuclease deficient FEN-1 can be a source of genomic instability in mammalian cells and an early step of carcinogenesis. In addition, we plan to investigate the cellular responses to the mutator phenotypes induced by these two replication mutators. Specific experiments proposed in this application are: 1. To use a yeast genetic screen to identify dominant mutant alleles in a specific region of Polalpha protein that have been show to play a significant role in genome stability maintenance. 2. To test the hypothesis that overexpression of a mammalian Polalpha mutant that has the homologous mutant allele that is identified above in yeast and overexpression of a nuclease- deficient FEN-1 mutant can induce a mutator phenotype in mammalian cells. 3. To analyze the cellular responses induced by the Polalpha mutants and by the nuclease deficient FEN-1 mutants to test the proposed mechanisms of generating the deletion and duplication imitator phenotypes. Results from these studies will contribute to our understanding of one of the molecular bases underlying the mechanisms that initiate the genomic instability and carcinogenesis.